The primary physiological problems in cystic fibrosis (CF) include abnormalities in epithelial chloride secretion, altered glycoprotein metabolism, and infections of the lung. In the majority of CF cases, these varied problems are the result of the deletion of a phenylalanine residue at position 508 in the recently identified Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), an integral membrane protein which appears to be a cyclic AMP-regulated chloride channel. Although we are now beginning to understand the physiological function of the CFTR, a cure for CF will probably only be realized when a suitable means for supplying copies of the correct CFTR to CF cells is worked out. The goal of the proposed studies is to develop effective techniques for introducing functional CFTR protein into CF cells. Specifically, we shall 1) further improve available CFTR expression systems to maximize the amount of CFTR that can be readily obtained, 2) attempt to develop a CFTR ATPase assay for the functionality of the CFTR, 3) develop a suitable procedure for purifying the CFTR in reasonably large amounts, 4) develop several procedures for reconstituting the purified CFTR into artificial lipid vesicles, 5) characterize the transmembrane orientation and functionality of the CFTR molecules in the reconstituted proteoliposomes, and 6) explore a variety of possible means for introducing the purified, reconstituted CFTR into cultured CF cells. The CFTR delivery experiments will involve a systematic biochemical approach to establish effective systems for incorporating the CFTR into the plasma membrane in the proper orientation with a reasonable half-life, followed by assessments of correction of the CF defect. If successful, these studies could provide the necessary methodology for future attempts to cure transgenic mice carrying the CF defect, and ultimately, CF patients.